RNA labeling method

ABSTRACT

Methods are described in which a sample containing RNA is contacted with an enzyme having an RNA ligation activity in the presence of a labeled substrate to provide labeled RNA. Methods of performing an array analysis of a labeled RNA sample are also described.

FIELD OF THE INVENTION

The invention relates generally to methods of biochemical analysis. Morespecifically, the invention relates to providing a method of attachingan observable label to RNA.

BACKGROUND OF THE INVENTION

Straightforward and reliable methods for simultaneously analyzingseveral constituents of a complex sample are extremely desirable.Polynucleotide arrays (such as DNA or RNA arrays) are known and areused, for example, as diagnostic or screening tools. Such arrays includeregions of usually different sequence polynucleotides (“capture agents”)arranged in a predetermined configuration on a support. The arrays are“addressable” in that these regions (sometimes referenced as “arrayfeatures”) have different predetermined locations (“addresses”) on thesupport of array. The polynucleotide arrays typically are fabricated onplanar supports either by depositing previously obtained polynucleotidesonto the support in a site specific fashion or by site specific in situsynthesis of the polynucleotides upon the support. After depositing thepolynucleotide capture agents onto the support, the support is typicallyprocessed (e.g., washed and blocked for example) and stored prior touse.

In use, an array is contacted with a sample or labeled sample containinganalytes (typically, but not necessarily, other polynucleotides) underconditions that promote specific binding of the analytes in the sampleto one or more of the capture agents present on the array. Thus, thearrays, when exposed to a sample, will undergo a binding reaction withthe sample and exhibit an observed binding pattern. This binding patterncan be detected upon interrogating the array. For example all targetpolynucleotides (for example, DNA) in the sample can be labeled with asuitable label (such as a fluorescent compound), and the label then canbe accurately observed (such as by observing the fluorescence pattern)on the array after exposure of the array to the sample. Assuming thatthe different sequence polynucleotides were correctly deposited inaccordance with the predetermined configuration, then the observedbinding pattern will be indicative of the presence and/or concentrationof one or more components of the sample. Techniques for scanning arraysare described, for example, in U.S. Pat. No. 5,763,870 and U.S. Pat. No.5,945,679. Still other techniques useful for observing an array aredescribed in U.S. Pat 5,721,435.

There has been great interest in the analysis of small RNAs, such asshort interfering RNAs (siRNAs), microRNAs (miRNA), tiny non-codingRNAs(tncRNA) and small modulatory RNA (smRNA), since the discovery of siRNAbiological activity over a decade ago. See Novina et al., Nature 430:161-164 (2004). Even though the functions of most discovered miRNAsremain a mystery, it has become clear that they exist in abundance inplants and animals, with up to tens of thousands of copies per cell. Inthe fruit fly, 78 have been identified, and over 200 have beenidentified in human (see the public database accessible via the websitelocated at >>http://www.sanger.ac.uk/cgi-bin/Rfam/mirna/browse.pl<<).The levels of individual miRNAs seem to vary with developmental stagesand tissue types. The level of fluctuation may be correlated withphenotype, mRNA levels, or protein levels for better biological insight.Thus quantitative measurements of miRNA may be of great importance.Further, viral miRNAs have been identified and may play a role inlatency (see Pfeffer et al., Science, 304: 734-736 (2004)), making thedetection and quantification of miRNAs a potentially valuable diagnostictool.

Analytic methods employing polynucleotide arrays have been used forinvestigating these small RNAs, e.g. miRNAs have become a subject ofinvestigation with microarray analysis. See, e.g., Liu et al., Proc.Nat'l Acad. Sci. USA, 101: 9740-9744 (2004); Thomson et al., NatureMethods, 1: 1-7 (2004); and Babak et al., RNA, 10: 1813-1819 (2004).Methods of labeling RNAs are of interest for use in array analysis ofRNA to provide an observable label used in interrogating the array. Inthe study of Liu et al., the miRNA was transcribed into DNA with abiotin-labeled primer. This primer was subsequently labeled withstreptavidin-linked Alexa dye prior to array hybridization. This methodis susceptible to any reverse-transcriptase reaction bias. Further, thestreptavidin-dye as well as streptavidin-biotin-RNA stochiometry may bedifficult to quantify. In the study of Thomson et al., the miRNA wasdirectly labeled with 5′-phosphate-cytidyl-uridyl-Cy3-3′ using T4 RNAligase. This reaction is sensitive to the acceptor sequence. See Englandet al., Biochemistry, 17: 2069-2776 (1978). In the study of Babak et al(4), the miRNA was labeled with Ulysis Alexa Fluor system, which reactswith guanine residue (G) of RNA. Since different miRNAs do not haveuniform G content, this method is not quantitative.

Thus, there is a continuing need for methods of labeling RNA with anobservable label. Such methods may be used in conjunction withanalytical methods based on observing the label, such as array-basedanalysis of polynucleotides.

SUMMARY OF THE INVENTION

The invention thus relates to novel methods for labeling RNA in asample. In particular embodiments, the invention provides methods inwhich a sample containing RNA is contacted with an enzyme having an RNAligation activity in the presence of a labeled substrate. This is doneunder conditions sufficient to result in coupling of the labeledsubstrate to the RNA in the sample to provide labeled RNA, theconditions including a DMSO concentration in the range from about 20% toabout 30%. The labeled substrate includes an observable label moietyattached to a nucleotide moiety.

Methods of performing an array analysis of an RNA sample are also taughtherein. In certain embodiments, the invention provides a method ofperforming an array analysis wherein the method includes labeling theRNA in the sample to provide labeled RNA. The labeled RNA is thencontacted with an array under conditions sufficient to provide forspecific binding of labeled RNA to the array. The array typically isthen interrogated to provide data on binding of RNA in the sample to thearray.

Additional objects, advantages, and novel features of this inventionshall be set forth in part in the descriptions and examples that followand in part will become apparent to those skilled in the art uponexamination of the following specifications or may be learned by thepractice of the invention. The objects and advantages of the inventionmay be realized and attained by means of the instruments, combinations,compositions and methods particularly pointed out in the appendedclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will be understood from thedescription of representative embodiments of the method herein and thedisclosure of illustrative apparatus for carrying out the method, takentogether with the Figures, wherein

FIG. 1 schematically illustrates embodiments of the present invention.

FIG. 2 schematically illustrates other embodiments of the presentinvention.

To facilitate understanding, identical reference numerals have beenused, where practical, to designate corresponding elements that arecommon to the Figures. Figure components are not drawn to scale.

DETAILED DESCRIPTION

Before the invention is described in detail, it is to be understood thatunless otherwise indicated this invention is not limited to particularmaterials, reagents, reaction materials, manufacturing processes, or thelike, as such may vary. It is also to be understood that the terminologyused herein is for purposes of describing particular embodiments only,and is not intended to be limiting. It is also possible in the presentinvention that steps may be executed in different sequence where this islogically possible. However, the sequence described below is preferred.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “an insoluble support” includes a plurality of insolublesupports. Similarly, reference to “an RNA” includes a plurality ofdifferent identity (sequence) RNA species.

Furthermore, where a range of values is provided, it is understood thatevery intervening value, between the upper and lower limit of that rangeand any other stated or intervening value in that stated range isencompassed within the invention. Also, it is contemplated that anyoptional feature of the inventive variations described may be set forthand claimed independently, or in combination with any one or more of thefeatures described herein. It is further noted that the claims may bedrafted to exclude any optional element. As such, this statement isintended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only,” and the like in connection with therecitation of claim elements, or use of a “negative” limitation. In thisspecification and in the claims that follow, reference will be made to anumber of terms that shall be defined to have the following meaningsunless a contrary intention is apparent.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.For example, if a step of a process is optional, it means that the stepmay or may not be performed, and, thus, the description includesembodiments wherein the step is performed and embodiments wherein thestep is not performed (i.e. it is omitted).

An “oligonucleotide” is a molecule containing from 2 to about 100nucleotide subunits. The term “nucleic acid” and “polynucleotide” areused interchangeably herein to describe a polymer of any length composedof nucleotides, e.g., deoxyribonucleotides or ribonucleotides, orcompounds produced synthetically (e.g., PNA as described in U.S. Pat.No. 5,948,902 and the references cited therein) which can hybridize withnaturally occurring nucleic acids in a sequence specific manneranalogous to that of two naturally occurring nucleic acids, e.g., canparticipate in Watson-Crick base pairing interactions. The terms“nucleoside” and “nucleotide” are intended to include those moietiesthat contain not only the known purine and pyrimidine bases, but alsoother heterocyclic bases that have been modified. Such modificationsinclude methylated purines or pyrimidines, acylated purines orpyrimidines, alkylated riboses or other heterocycles. In addition, theterms “nucleoside” and “nucleotide” include those moieties that containnot only conventional ribose and deoxyribose sugars, but other sugars aswell. Modified nucleosides or nucleotides also include modifications onthe sugar moiety, e.g., wherein one or more of the hydroxyl groups arereplaced with halogen atoms or aliphatic groups, or are functionalizedas ethers, amines, or the like. “Analogues” refer to molecules havingstructural features that are recognized in the literature as beingmimetics, derivatives, having analogous structures, or other like terms,and include, for example, polynucleotides incorporating non-natural (notusually occurring in nature) nucleotides, unnatural nucleotide mimeticssuch as 2′-modified nucleosides, peptide nucleic acids, oligomericnucleoside phosphonates, and any polynucleotide that has addedsubstituent groups, such as protecting groups or linking moieties.

“Moiety” and “group” are used to refer to a portion of a molecule,typically having a particular functional or structural feature, e.g. alinking group (a portion of a molecule connecting two other portions ofthe molecule), or an ethyl moiety (a portion of a molecule with astructure closely related to ethane). A moiety is generally bound to oneor more other moieties to provide a molecular entity. As a simpleexample, a hydroxyl moiety bound to an ethyl moiety provides an ethanolmolecule. At various points herein, the text may refer to a moiety bythe name of the most closely related structure (e.g. an oligonucleotidemoiety may be referenced as an oligonucleotide, a mononucleotide moietymay be referenced as a mononucleotide). However, despite this seeminginformality of terminology, the appropriate meaning will be clear tothose of ordinary skill in the art given the context, e.g. if thereferenced term has a portion of its structure replaced with anothergroup, then the referenced term is usually understood to be the moiety.For example, a mononucleotide moiety is a single nucleotide which has aportion of its structure (e.g. a hydrogen atom, hydroxyl group, or othergroup) replaced by a different moiety (e.g. a linking group, anobservable label moiety, or other group). Similarly, an oligonucleotidemoiety is an oligonucleotide which has a portion of its structure (e.g.a hydrogen atom, hydroxyl group, or other group) replaced by a differentmoiety (e.g. a linking group, an observable label moiety, or othergroup). “Nucleotide moiety” is generic to both mononucleotide moiety andoligonucleotide moiety.

“Linkage” as used herein refers to a first moiety bonded to two othermoieties, wherein the two other moieties are linked via the firstmoiety. Typical linkages include ether (—O—), oxo (—C(O)—), amino(—NH—), amido (—N—C(O)—), thio (—S—), phospho (—P—), ester (—O—C(O)—).

“Bound” may be used herein to indicate direct or indirect attachment. Inthe context of chemical structures, “bound” (or “bonded”) may refer tothe existence of a chemical bond directly joining two moieties orindirectly joining two moieties (e.g. via a linking group or any otherintervening portion of the molecule). The chemical bond may be acovalent bond, an ionic bond, a coordination complex, hydrogen bonding,van der Waals interactions, or hydrophobic stacking, or may exhibitcharacteristics of multiple types of chemical bonds. In certaininstances, “bound” includes embodiments where the attachment is directand also embodiments where the attachment is indirect. “Free,” as usedin the context of a moiety that is free, indicates that the moiety isavailable to react with or be contacted by other components of thesolution in which the moiety is a part.

“Isolated” or “purified” generally refers to isolation of a substance(compound, polynucleotide, protein, polypeptide, polypeptide,chromosome, etc.) such that the substance comprises a substantialportion of the sample in which it resides (excluding solvents), i.e.greater than the substance is typically found in its natural orun-isolated state. Typically, a substantial portion of the samplecomprises at least about 5%, at least about 10%, at least about 20%, atleast about 30%, at least about 50%, preferably at least about 80%, ormore preferably at least about 90% of the sample (excluding solvents).For example, a sample of isolated RNA will typically comprise at leastabout 5% total RNA, where percent is calculated in this context as mass(e.g. in micrograms) of total RNA in the sample divided by mass (e.g. inmicrograms) of the sum of (total RNA+other constituents in the sample(excluding solvent). Techniques for purifying polynucleotides andpolypeptides of interest are well known in the art and include, forexample, gel electrophresis, ion-exchange chromatography, affinitychromatography, flow sorting, and sedimentation according to density. Intypical embodiments, one or more of the sample, the enzyme having an RNAligation activity, and the labeled substrate is in isolated form; moretypically, all three are obtained in isolated form prior to use in thepresent methods.

The term “sample” as used herein relates to a material or mixture ofmaterials, typically, although not necessarily, in fluid form,containing one or more components of interest.

The term “analyte” is used herein to refer to a known or unknowncomponent of a sample. In certain embodiments of the invention, ananalyte may specifically bind to a capture agent on a support surface ifthe analyte and the capture agent are members of a specific bindingpair. In general, analytes are typically RNA or other polynucleotides.Typically, an “analyte” is referenced as a species in a mobile phase(e.g., fluid), to be detected by a “capture agent” which, in someembodiments, is bound to a support, or in other embodiments, is insolution. However, either of the “analyte” or “capture agent” may be theone which is to be evaluated by the other (thus, either one could be anunknown mixture of components of a sample, e.g., polynucleotides, to beevaluated by binding with the other). A “target” references an analyte.

The term “capture agent” refers to an agent that binds an analytethrough an interaction that is sufficient to permit the agent to bindand concentrate the analyte from a homogeneous mixture of differentanalytes. The binding interaction may be mediated by an affinity regionof the capture agent. Representative capture agents include polypeptidesand polynucleotides, for example antibodies, peptides, or fragments ofdouble stranded or single-stranded DNA or RNA may employed. Captureagents usually “specifically bind” one or more analytes.

The term “specific binding” refers to the ability of a capture agent topreferentially bind to a particular analyte that is present in ahomogeneous mixture of different analytes. In certain embodiments, aspecific binding interaction will discriminate between desirable andundesirable analytes in a sample, in some embodiments more than about 10to 100-fold or more (e.g., more than about 1000- or 10,000-fold). Incertain embodiments, the binding constant of a capture agent and analyteis greater than 10⁶ M⁻¹, greater than 10⁷ M⁻¹, greater than 10⁸ M⁻¹,greater than 10⁹ M⁻¹, greater than 10¹⁰ M⁻¹, usually up to about 10¹²M⁻¹, or even up to about 10¹⁵ M⁻¹.

The term “stringent assay conditions” as used herein refers toconditions that are compatible to produce binding pairs of nucleicacids, e.g., capture agents and analytes, of sufficient complementarityto provide for the desired level of specificity in the assay while beingincompatible to the formation of binding pairs between binding membersof insufficient complementarity to provide for the desired specificity.Stringent assay conditions are the summation or combination (totality)of both hybridization and wash conditions.

A “stringent hybridization” and “stringent hybridization washconditions” in the context of nucleic acid hybridization (e.g., as inarray, Southern or Northern hybridizations) are sequence dependent, andare different under different experimental conditions. Stringenthybridization conditions that can be used to identify nucleic acidswithin the scope of the invention can include, e.g., hybridization in abuffer comprising 50% formamide, 5×SSC, and 1% SDS at 42° C., orhybridization in a buffer comprising 5×SSC and 1% SDS at 65° C., bothwith a wash of 0.2×SSC and 0.1% SDS at 65° C. Exemplary stringenthybridization conditions can also include a hybridization in a buffer of40% formamide, 1 M NaCl, and 1% SDS at 37° C., and a wash in 1×SSC at45° C. Alternatively, hybridization to filter-bound DNA in 0.5 M NaHPO4,7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in0.1×SSC/0.1% SDS at 68° C. can be employed. Yet additional stringenthybridization conditions include hybridization at 60° C. or higher and3×SSC (450 mM sodium chloride/45 mM sodium citrate) or incubation at 42°C. in a solution containing 30% formamide, 1M NaCl, 0.5% sodiumsarcosine, 50 mM MES, pH 6.5. Those of ordinary skill will readilyrecognize that alternative but comparable hybridization and washconditions can be utilized to provide conditions of similar stringency.

In certain embodiments, the stringency of the wash conditions may affectthe degree to which nucleic acids are specifically hybridized tocomplementary capture agents. Wash conditions used to identify nucleicacids may include, e.g.: a salt concentration of about 0.02 molar at pH7 and a temperature of at least about 50° C. or about 55° C. to about60° C.; or, a salt concentration of about 0.15 M NaCl at 72° C. forabout 15 minutes; or, a salt concentration of about 0.2×SSC at atemperature of at least about 50° C. or about 55° C. to about 60° C. forabout 1 to about 20 minutes; or, multiple washes with a solution with asalt concentration of about 0.1×SSC containing 0.1% SDS at 20 to 50° C.for 1 to 15 minutes; or, equivalent conditions. Stringent conditions forwashing can also be, e.g., 0.2×SSC/0.1% SDS at 42° C. In instanceswherein the nucleic acid molecules are deoxyoligonucleotides (i.e.,oligonucleotides), stringent conditions can include washing in6×SSC/0.05% sodium pyrophosphate at 37° C. (for 14-base oligos), 48° C.(for 17-base oligos), 55° C. (for 20-base oligos), and 60° C. (for23-base oligos). See Sambrook, Ausubel, or Tijssen (cited below) fordetailed descriptions of equivalent hybridization and wash conditionsand for reagents and buffers, e.g., SSC buffers and equivalent reagentsand conditions.

A specific example of stringent assay conditions is rotatinghybridization at a temperature of about 55° C. to about 70° C. in a saltbased hybridization buffer with a total monovalent cation concentrationof 1.5M (e.g., as described in U.S. patent application Ser. No.09/655,482 filed on Sep. 5, 2000, the disclosure of which is hereinincorporated by reference) followed by washes of 0.5×SSC and 0.1×SSC atroom temperature and 37° C.

Stringent hybridization conditions may also include a “prehybridization”of aqueous phase nucleic acids with complexity-reducing nucleic acids tosuppress repetitive sequences. For example, certain stringenthybridization conditions include, prior to any hybridization tosurface-bound polynucleotides, hybridization with Cot-1 DNA or withrandom sequence synthetic oligonucleotides (e.g. 25-mers), or the like.

Stringent assay conditions are hybridization conditions that are atleast as stringent as the above representative conditions, where a givenset of conditions are considered to be at least as stringent ifsubstantially no additional binding complexes that lack sufficientcomplementarity to provide for the desired specificity are produced inthe given set of conditions as compared to the above specificconditions, where by “substantially no more” is meant less than about5-fold more, typically less than about 3-fold more. Other stringenthybridization conditions are known in the art and may also be employed,as appropriate.

The term “pre-determined” refers to an element whose identity is knownprior to its use. For example, a “pre-determined analyte” is an analytewhose identity is known prior to any binding to a capture agent. Anelement may be known by name, sequence, molecular weight, its function,or any other attribute or identifier. In some embodiments, the term“analyte of interest”, i.e., a known analyte that is of interest, isused synonymously with the term “pre-determined analyte”.

The term “array” encompasses the term “microarray” and refers to anordered array of capture agents for binding to aqueous analytes and thelike. An “array” includes any two-dimensional or substantiallytwo-dimensional (as well as a three-dimensional) arrangement ofspatially addressable regions (i.e., “features”) containing captureagents, particularly polynucleotides, and the like. Any given supportmay carry one, two, four or more arrays disposed on a surface of asupport. Depending upon the use, any or all of the arrays may be thesame or different from one another and each may contain multiple spotsor features. A typical array may contain one or more, including morethan two, more than ten, more than one hundred, more than one thousand,more ten thousand features, or even more than one hundred thousandfeatures, in an area of less than 100 cm², 20 cm² or even less than 10cm², e.g., less than about 5 cm²,including less than about 1 cm², lessthan about 1 mm², e.g., 100 μm², or even smaller. For example, featuresmay have widths (that is, diameter, for a round spot) in the range froma 10 μm to 1.0 cm. In other embodiments each feature may have a width inthe range of 1.0 μm to 1.0 mm, usually 5.0 μm to 500 μm, and moreusually 10 μm to 200 μm. Non-round features may have area rangesequivalent to that of circular features with the foregoing width(diameter) ranges. At least some, or all, of the features are of thesame or different compositions (for example, when any repeats of eachfeature composition are excluded the remaining features may account forat least 5%, 10%, 20%, 50%, 95%, 99% or 100% of the total number offeatures). Inter-feature areas will typically (but not essentially) bepresent which do not carry any nucleic acids (or other biopolymer orchemical moiety of a type of which the features are composed). Suchinter-feature areas typically will be present where the arrays areformed by processes involving drop deposition of reagents but may not bepresent when, for example, photolithographic array fabrication processesare used. It will be appreciated though, that the inter-feature areas,when present, could be of various sizes and configurations.

Arrays can be fabricated by depositing (e.g., by contact- or jet-basedmethods) either precursor units (such as nucleotide or amino acidmonomers) or pre-synthesized capture agent. An array is “addressable”when it has multiple regions of different moieties (e.g., differentcapture agent) such that a region (i.e., a “feature” or “spot” of thearray) at a particular predetermined location (i.e., an “address”) onthe array will detect a particular sequence. An “array layout” refers toone or more characteristics of the features, such as feature positioningon the support, one or more feature dimensions, and an indication of amoiety at a given location. “Interrogating” the array refers toobtaining information from the array, especially information aboutanalytes binding to the array. “Hybridization assay” references aprocess of contacting an array with a mobile phase containing analyte.An “array support” refers to an article that supports an addressablecollection of capture agents.

“Complementary” references a property of specific binding betweenpolynucleotides based on the sequences of the polynucleotides. As usedherein, polynucleotides are complementary if they bind to each other ina hybridization assay under stringent conditions, e.g. if they produce agiven or detectable level of signal in a hybridization assay. Portionsof polynucleotides are complementary to each other if they followconventional base-pairing rules, e.g. A pairs with T (or U) and G pairswith C. “Complementary” includes embodiments in which there is anabsolute sequence complementarity, and also embodiments in which thereis a substantial sequence complementarity. “Absolute sequencecomplementarity” means that there is 100% sequence complementaritybetween a first polynucleotide and a second polynucleotide, i.e. thereare no insertions, deletions, or substitutions in either of the firstand second polynucleotides with respect to the other polynucleotide(over the complementary region). Put another way, every base of thecomplementary region may be paired with its complementary base, i.e.following normal base-pairing rules. “Substantial sequencecomplementarity” permits one or more relatively small (less than 10bases, e.g. less than 5 bases, typically less than 3 bases, moretypically a single base) insertions, deletions, or substitutions in thefirst and/or second polynucleotide (over the complementary region)relative to the other polynucleotide. The region that is complementarybetween a first polynucleotide and a second polynucleotide (e.g. atarget analyte and a capture agent) is typically at least about 10 baseslong, more typically at least about 15 bases long, still more typicallyat least about 20 bases long, or at least about 25 bases long. Invarious typical embodiments, the region that is complementary between afirst polynucleotide and a second polynucleotide (e.g. target analyteand a capture agent) may be up to about 200 bases long, or up to about120 bases long, up to about 100 bases long, up to about 80 bases long,up to about 60 bases long, or up to about 45 bases long.

“Upstream” as used herein refers to the 5′ direction along apolynucleotide, e.g. an RNA molecule. “Downstream” refers to the 3′direction along the polynucleotide. Hence, a label downstream of ananalyte is located at (or is bound to) a nucleotide moiety that islocated in the 3′ direction from the analyte, e.g. bound to the 3′ endof the analyte. Similarly, an “upstream label” references a label thatis located at (or is bound to) a nucleotide moiety that is located inthe 5′ direction from the analyte, e.g. bound to the 5′ end of theanalyte. “3′-” and “5′-” have their conventional meaning as known in theart. A 5′-phosphate is a phosphate group located at the 5′-end of apolynucleotide. A 3′-hydroxyl is a hydroxyl group located at the 3′-endof a polynucleotide. As an example, FIG. 1 illustrates downstreamlabeling of an analyte. As another example, FIG. 2 illustrates upstreamlabeling of an analyte. If the polynucleotide is double stranded, one ofthe strands is selected as the reference strand, e.g. the strand that islabeled, or the strand that is not labeled (or some other criteria orfeature of the strand may be used to designate one strand as thereference strand).

Accordingly, in one embodiment of the present invention, a method oflabeling RNA in a sample is provided. The method includes contacting thesample with an enzyme having an RNA ligation activity in the presence ofa labeled substrate under conditions sufficient to result in coupling ofthe labeled substrate to the RNA in the sample to provide labeled RNA,the conditions including a DMSO concentration in the range from about20% to about 30%, wherein the labeled substrate comprises an observablelabel moiety attached to a nucleotide moiety.

The sample may be any RNA sample, typically a sample containing RNA thathas been isolated from a biological source, e.g. any plant, animal,yeast, bacterial, or viral source, or a non-biological source, e.g.chemically synthesized. The dimethylsulfoxide (DMSO) concentration iscalculated as volume (e.g. in milliliters) of DMSO divided by totalvolume (e.g. in milliliters) of the solution containing the DMSO. Thisquantity is typically cast as a percentage by multiplying by 100%. Forexample, the DMSO concentration will be in a range of 20% to about 30%,calculated as the volume of DMSO in the solution resulting fromcontacting the sample with an enzyme having an RNA ligation activity inthe presence of a labeled substrate, divided by the total volume of thesolution, and then multiplying by 100%. The other components present inthe resulting solution will typically be water, buffer components, salt,RNA, labeled substrate, and enzyme having an RNA ligation activity,although other components may also be present. In particularembodiments, the sample includes small RNAs, especially RNAs less thanabout 500 bases long, e.g. less than about 400 bases long, less thanabout 300 bases long, less than about 200 bases long, or less than about100 bases long. In particular embodiments, the sample includes one ormore short RNAs, such as e.g. short interfering RNAs (siRNAs), microRNAs(miRNA), tiny non-coding RNAs (tncRNA) and small modulatory RNA (smRNA).See Novina et al., Nature 430: 161-164 (2004). In particularembodiments, the sample includes isolated small RNAs, e.g. the sampleresults from an isolation protocol for small RNA such as one or more ofthose listed in this paragraph. In certain embodiments, the small RNAtargets may include isolated miRNAs, such as those described in theliterature and in the public database accessible via the website locatedat >>http://www.sanger.ac.uk/cgi-bin/Rfam/mirna/browse.pl<<. Inparticular embodiments, the sample includes isolated small RNAs, e.g.the sample results from an isolation protocol for small RNA, especiallyRNAs less than about 500 bases long, e.g. less than about 400 baseslong, less than about 300 bases long, less than about 200 bases long,less than about 100 bases long, or less than about 50 bases long.

The enzyme having an RNA ligation activity is typically any RNA ligaseenzyme, although other enzymes capable of coupling the labeled substrateto the RNA may be used. In particular embodiments, the enzyme having anRNA ligation activity is capable of coupling a nucleotide (oroligonucleotide, or RNA) having a 5′ phosphate to an oligonucleotidehaving a 3′ hydroxyl. Exemplary enzymes include T4 RNA ligase availablefrom Amersham/Pharmacia company, ThermoPhage™ RNA ligase II (availablefrom Prokaria LTD, Iceland), or other available RNA ligase enzymes knownto be capable of coupling a nucleotide (or oligonucleotide, or RNA)having a 5′ phosphate to an oligonucleotide having a 3′ hydroxyl. Incertain embodiments, the enzyme may be selected from yeast poly Apolymerase, E. coli poly A polymerase, or terminal transferase (each ofwhich is available from Amersham/Pharmacia). The enzyme having an RNAligation activity should be selected such that the enzyme is capableperforming the coupling when one (or both) of the nucleotide (oroligonucleotide) having a 5′phosphate and/or the oligonucleotide havinga 3′ hydroxyl includes a label. Selection of the enzyme having an RNAligation activity will typically be based on availability of the enzymeand activity of the enzyme under the desired reaction conditions for thecoupling (e.g. temperature, pH, ionic strength, source of RNA and/orlabeled substrate, structural feature of RNA and/or labeled substrate,concentration of RNA and/or labeled substrate, presence of othermaterials (e.g. contaminants, salt, surfactant, other solvents) etc.)

The coupling reaction is conducted under conditions sufficient to resultin coupling. The conditions of the coupling reaction will generally beselected with regard to the known (previously described) conditions foruse of the particular enzyme chosen for use in the methods of theinvention, with the specific modifications described herein. As alreadyindicated, the DMSO of the reaction mixture for the coupling reactionwill be in the range of 20% to 30%. Other experimental parameters may beselected based on known ranges for the experimental parameters ordetermined through routine experimentation based on, e.g. efficacy ofthe labeling reaction. Such other experimental parameters may include,e.g. temperature, pH, ionic strength, source of RNA and/or labeledsubstrate, structural feature of RNA and/or labeled substrate,concentration of RNA and/or labeled substrate, presence of othermaterials (e.g. contaminants, salt, surfactant, other solvents) etc.

The labeled substrate comprises an observable label moiety attached to anucleotide moiety. The nucleotide moiety is typically a mononucleotidemoiety or an oligonucleotide moiety. In particular embodiments, thenucleotide moiety is less than about 100 bases long. In embodimentswhich result in upstream labeling of the RNA (examples shown in FIG. 2),the nucleotide moiety will typically be at least three bases long. Incertain embodiments, the nucleotide moiety will be less than 50 baseslong, e.g. less than 40 bases long, less than 30 bases long, less than20 bases long. In some embodiments, the nucleotide moiety will be 1, 2,3, 4, 5, or 6 bases long. The nucleotide moiety may typically have anydesired sequence or even an unknown sequence. In certain embodiments, aplurality of labeled substrates may be used in the same reaction (e.g. aplurality of nucleotide moieties, each having a different sequence, eachhaving an observable label moiety attached), thereby resulting inligating one of a plurality of nucleotide moieties to each molecule ofRNA.

The observable label moiety is a moiety that provides for an observablesignal that indicates the presence of the observable label moiety.Typical examples include a chromogenic moiety, a fluorophore, a masslabel, a spin label, a radiolabel, or other labels known in the art. Inparticular embodiments, the observable label moiety is a fluorophoreselected from the group consisting of Cy3, Cy5, and an Alexa dye.Further examples of label moieties include any commercially availablefluorophores that can be conjugated to mononucleotides orpolynucleotides, e.g. dyes from Molecular Probes (Eugene, Oreg. andLeiden, The Netherlands) such as the Alexa Fluor series (example: Alexa350, Alexa 430, Alexa 532, Alexa 546, Alexa 568, and Alexa 594) and theseries of BODIPY conjugates. Other examples include: Tamra, Fluorescein,carboxyfluorescene, JOE, rhodamine, carboxyrhodamine, CY series, Oysterseries. More information about commercially available dyes foroligonucleotide conjugation can be found at the website locatedat >>http://www.synthegen.com<<. Any such dyes may potentially be usedin accordance with the methods described herein. Although the examplesdescribed herein use a fluorophore as the label, it will be apparent tothose of ordinary skill that other labels may be used (instead of afluorophore, or even in addition to a fluorophore). Such labelstypically are well known in the art.

The observable label moiety may be attached to the nucleotide moiety atany site of the nucleotide moiety that is compatible with the ligationreaction. In other words, the label moiety should not prevent theligation reaction, e.g. by interfering with the enzyme having an RNAligation activity. In embodiments of downstream labeling (such asillustrated in FIG. 1), the label moiety may be attached to thenucleotide moiety via the 3′-site at the end of the nucleotide moiety ormay be attached at any available “internal” site (i.e. other than the3′-site or 5′-site) of the nucleotide moiety. In embodiments of upstreamlabeling (such as illustrated in FIG. 2), the label moiety may beattached to the nucleotide moiety via the 5′-site at the end of thenucleotide moiety or may be attached at any available “internal” site(i.e. other than the 3′-site or 5′-site) of the nucleotide moiety. Theobservable label moiety could be incorporated as a specialphosphoramidite during the oligoribonucleotide synthesis or as apost-synthetic modification. An example of the phosphoramidite methodincludes direct coupling of label-containing phosphoramidite duringsynthesis of the oligonucleotide moiety, or the incorporation ofamino-activated phosphoramidite during synthesis of the oligonucleotidemoiety, which enables post-synthetic coupling to desired observablelabel moiety. In particular embodiments, the observable label moiety isattached to the nucleotide moiety via a linking group, wherein thelinking group may be attached to the nucleotide moiety via a 5′ terminusor a 3′ terminus of the nucleotide moiety or any other available site ofthe nucleotide moiety. The linking group may be any linking group knownin the art that does not prevent the ligation reaction (e.g. does notprevent the enzyme having an RNA ligation activity from ligating the RNAand the labeled substrate). Any such linking groups or other means ofattachment of the label moiety to the nucleotide moiety known in the artmay provide for the labeled substrate.

Thus, in particular embodiments, the labeled substrate has thestructure:N-D

wherein: N is selected from a mononucleotide moiety or anoligonucleotide moiety having a length of less than about 100 bases, and

-   -   D is an observable label moiety (e.g. attached to N via a 5′        terminus or a 3′ terminus of N, or any other available site of        N).

An embodiment of a method in accordance with the present invention isillustrated in FIG. 1. In FIG. 1, the method 100 of labeling RNAincludes adding 104 DMSO 106 to the sample (which includes the RNA 102).The labeled substrate 110A, 110B or 110C may then be added 108 to theresulting solution (containing the DMSO 106 and the RNA 102 from thesample). The labeled substrate 110A, 110B or 110C typically is amononucleotide with a 3′ fluorophore and 5′ phosphate 110A, anoligonucleotide with a 5′ phosphate and one or more internalfluorophore(s) 110B, or an oligonucleotide with a 3′ fluorophore and 5′phosphate 110C. (In an embodiment in which the labeled substrate 110Bhas one or more internal fluorophores, the 3′ end of the oligonucleotidelacks the 3′ hydroxyl, as indicated by the ‘X’ in FIG. 1. For example, aperiodate oxidation reaction may be used to modify the 3′ end, or the 3′end may be modified by having a fluorophore bound thereto.) The enzymehaving an RNA ligation activity 114 is also added 112. In typicalembodiments, the concentrations of the solutions and the volumes addedare planned to provide that the resulting solution has the desiredconcentration of DMSO (e.g. in the range of about 20% to about 30%, moretypically in the range of about 22% to about 28%, even more typically inthe range of about 24% to about 26%). The resulting solution is thenallowed to react 116 under conditions and for a time sufficient for thecoupling of the labeled substrate to the RNA to occur, thereby providingthe labeled RNA 118A, 118B, or 118C. Typical conditions 120 of overnightincubation at 16° C. are shown for the embodiment of FIG. 1, althoughthese conditions may vary depending on the particular enzyme used andthe RNA and labeled substrate provided. In the illustrated embodiment,the label is a fluorophore 111, but other labels may be used as long asthe coupling of the labeled substrate to the RNA may still occur.Selection and optimization of the conditions is within routineexperimentation for one of ordinary skill in the art given thedisclosure herein.

Another embodiment of a method in accordance with the present inventionis illustrated in FIG. 2. In FIG. 2, the method 200 of labeling RNAincludes pre-treating the RNA 102 by performing a periodate oxidationreaction 222 on the initial RNA sample. The periodate oxidation reactionhelps prevent the RNA from concatenating or from ligating to itself whenthe enzyme having an RNA ligation activity is later added. The periodateoxidation reaction 222 is followed with inactivation and de-salting 224of the solution resulting from the periodate oxidation reaction. Theperiodate oxidation reaction may be performed using any protocol knownin the art. See, e.g. Neu, Harold C., et al., J. Biol. Chem 239: 2927-34(1964); Kurata, Shinya, et al., Nucleic Acids Res. 31: e145 (2003);England, T. E., et al., Meth. Enzymol. 65: 65-74 (1980); England, T. E.,et al., Nature 275: 560-61 (1978); and Maruyama, K., et al., Gene 138:171-74 (1994). In a typical periodate oxidation reaction,ribonucleotides are treated with estimated 5-fold molar excess of sodiummetaperiodate for 20 minutes to 1 hour at 0° C. to room temperature inthe dark. The ribonucleotides can be cleaned from the solution byethanol precipitation or desalting columns, such as BioRad MicroBio-Spin™ 6.

Continuing with the embodiment shown in FIG. 2, the method 200 includesadding 204 DMSO 206 to the solution containing the RNA 202. The labeledsubstrate 210A or 210B may then be added 208 to the resulting solution(containing the DMSO 206 and the RNA 202 from the sample). The labeledsubstrate 210A or 210B typically is an oligonucleotide with a 5′fluorophore 210A or an oligonucleotide with one or more internalfluorophore(s) 210B. The enzyme having an RNA ligation activity 214 isalso added 212. In typical embodiments, the concentrations of thesolutions and the volumes added are planned to provide that theresulting solution has the desired concentration of DMSO (e.g. in therange of about 20% to about 30%, more typically in the range of about22% to about 28%, even more typically in the range of about 24% to about26%). The resulting solution is then allowed to react 216 underconditions and for a time sufficient for the coupling of the labeledsubstrate to the RNA to occur, thereby providing the labeled RNA 218A or218B (products corresponding to 210A or 210B, respectively). Typicalconditions 220 of overnight incubation at 16° C. are shown for theembodiment of FIG. 2, although these conditions may vary depending onthe particular enzyme used and the RNA and labeled substrate provided.For example, the temperature of the incubation will typically be in therange from about 4° C. to about 37° C., more typically in the range fromabout 10° C. to about 30° C., still more typically in the range fromabout 14° C. to about 20° C. In the illustrated embodiment, the label isa fluorophore 211, but other labels may be used as long as the couplingof the labeled substrate to the RNA may still occur. Selection andoptimization of the conditions is within routine experimentation for oneof ordinary skill in the art given the disclosure herein.

In the embodiment illustrated in FIG. 1, the labeled substratedenominated 110A is shown as a mononucleotide attached to a fluorophore.In other embodiments illustrated in FIG. 1, the labeled substrates 110Band 110C are shown as an oligonucleotide having one or morefluorophore(s) attached to the oligonucleotide, in which theoligonucleotide does not have a 3′ hydroxyl. The lack of the 3′ hydroxylhelps prevent concatenation of the labeled substrate. This is analogousto the embodiment shown in FIG. 2, wherein the RNA 202 is subjected toperiodate oxidation to help prevent the RNA from concatenating or fromligating to itself. The labeled substrate 110C is shown as anoligonucleotide having a fluorophore(s) attached to an oligonucleotideat the 3′ end of the oligonucleotide. In FIG. 2, the labeled substratedenominated 210A and 210B illustrate representative embodiments in whichthe labeled substrate includes an oligonucleotide attached to one ormore fluorophores. In certain embodiments, the labeled substrateincludes an oligonucleotide having attached thereto a plurality, e.g. 2,3, 4, 5, or more label moieties, up to about 10, 20, 30 or more labelmoieties, attached to the oligonucleotide moiety at internal (notterminal) sites. In certain such embodiments, a label moiety is alsoattached to the oligonucleotide via one of the 3′ or 5′ terminal carbonsof the oligonucleotide.

In particular embodiments, the enzyme having an RNA ligation activitycatalyzes a coupling reaction between a donor molecule having a5′-phosphate and an acceptor molecule having a 3′-hydroxyl, as shown inthe reaction:

Where: Acc-3′-OH is the acceptor molecule having a 3′-hydroxyl;

-   -   PO₄-5′-Don is the donor molecule having a 5′-phosphate;    -   Acc-3′-O—PO₃-5′-Don is the product having the coupled donor and        acceptor moieties (e.g. the labeled RNA); and    -   (enz) is the enzyme having an RNA ligation activity.

In certain embodiments, such as that illustrated in FIG. 1, the acceptormolecule is the RNA 102 and the donor molecule is the labeled substrate110A, 110B or 110C. The resulting product 118A, 118B, or 118C has thelabeled substrate moiety downstream from the RNA, i.e. the product is adownstream labeled RNA. In other embodiments, such as that illustratedin FIG. 2, the acceptor molecule is the labeled substrate 210A or 210Band the donor molecule is the RNA 202. The resulting product 218A or218B has the labeled substrate moiety upstream from the RNA, i.e. theproduct is an upstream labeled RNA. Thus, based upon selection of theRNA and labeled substrate as disclosed herein, methods in accordancewith the present invention may result in either upstream or downstreamlabeled RNA.

It should be noted that the general utility of the method is not limitedto the particular sequence of steps shown in the figures. Othersequences of steps leading to essentially similar results are intendedto be included in the invention. For example, in certain embodiments,the labeled substrate may be dissolved in a solution that includes theDMSO, and the resulting solution mixed with the sample prior tocontacting with the enzyme having an RNA ligase activity. Thus, inparticular embodiments, the invention includes any process which resultsin contacting the sample with the enzyme having an RNA ligation activityin the presence of the labeled substrate under conditions which includea DMSO concentration in the range from about 20% to about 30%.

With reference to FIG. 1, in certain embodiments, after the DMSO 106 isadded 104 and before the enzyme having an RNA ligation activity 114 isadded 112, the method 100 includes heating the solution containing theDMSO 106 and the RNA 102 from the sample. In this optional heating step,the RNA is typically heated to a temperature of at least about 80° C.(e.g. at least about 85° C., at least about 90° C., at least about 95°C.; and up to about 105° C. or 110° C.) under conditions that include aDMSO concentration of at least about 40% DMSO (typically up to about 60%DMSO, although in some embodiments the DMSO concentration may be up to70% DMSO, up to 80% DMSO, or even more). This optional heating ismaintained for at least 10 seconds, typically at least about 20 seconds,at least about 30 seconds, at least about 1 minute, at least about 2minutes, and up to about 15 minutes, or more. In particular embodiments,reaction solutions of up to about 50 microliters are heated for about 30to about 60 seconds per 5-10 microliters of reaction solution. After theheating, the RNA is typically quickly cooled (e.g. to less than about40° C., more typically less than about 20° C., or in some embodimentsless than about 5° C.) before adding the enzyme having an RNA ligationactivity. With reference to FIG. 2, a similar heating step mayoptionally be included in embodiments such as that illustrated in thefigure.

It should be noted that, in particular embodiments, the RNA in thesample is isolated via a process that results in the RNA in the samplehaving a 5′-phosphate. For embodiments such as that pictured in FIG. 1,in which the RNA in the sample does not have a 5′-phosphate, apreparatory treatment of subjecting the RNA in the sample to adephosphorylation reaction is conducted prior to labeling the RNA in thesample by the ligation method illustrated in FIG. 1. Suchdephosphorylation reactions are well known in the art, for example,treating the RNA sample with an enzyme having a 5′-phosphatase activity,e.g. calf intestine alkaline phosphatase, shrimp alkaline phosphatase,or E. coli alkaline phosphatase, or any other method ofdephosphorylating the RNA known in the art. Thus, in certainembodiments, the method of labeling RNA in a sample includes, prior tocontacting the sample with the enzyme having an RNA ligation activity,contacting the sample with an enzyme having a 5′-phosphatase activity toremove 5′-phosphate groups from the RNA in the sample.

In some embodiments, the labeled substrate has only one observable labelmoiety attached to the nucleotide moiety. In such embodiments, thelabeled RNA will consist essentially of RNA labeled with a single labelmoiety (i.e. each labeled RNA molecule will have only one observablelabel moiety attached—referenced herein as “singly-labeled RNA”). Thispotentially provides increased ease of use in quantitative methods usingthe labeled RNA.

In other embodiments, the nucleotide moiety of the labeled substrate hasa plurality of observable label moieties. In such embodiments, when thelabeling reaction is performed to yield the labeled RNA, each labeledRNA molecule will have a plurality of observable label moieties(referenced herein as “multiply-labeled RNA”). Thus, the labeled RNAwill consist essentially of RNA labeled with a plurality of labelmoieties. This increased labeling of the RNA may provide for greatersensitivity in analyses using the labeled RNA. In particularembodiments, each labeled RNA molecule in the sample will be labeledwith a consistent number of observable label moieties (relative to theother labeled RNA molecules in the sample). This provides opportunityfor more quantitative analysis of labeled RNA than in methods thatprovide an inconsistent number of observable labels per labeled RNAmolecule.

In certain embodiments, methods of performing an array analysis of anRNA sample are provided. In certain embodiments, the invention providesa method of performing an array analysis wherein the method includeslabeling the RNA in the sample to provide labeled RNA using a labelingmethod in accordance with the methods described herein. The labeled RNAis then contacted with an array under conditions sufficient to providefor specific binding of labeled RNA to the array. The array typically isthen interrogated to provide data on binding of the labeled RNA to thearray.

Standard hybridization techniques (using stringent hybridizationconditions) are used to hybridize a labeled sample to a nucleic acidarray. Suitable methods are described in references describing CGHtechniques (Kallioniemi et al., Science 258:818-821 (1992) and WO93/18186). Several guides to general techniques are available, e.g.,Tijssen, Hybridization with Nucleic Acid Probes, Parts I and II(Elsevier, Amsterdam 1993). For descriptions of techniques suitable forin situ hybridizations, see Gall et al. Meth. Enzymol., 21:470-480(1981); and Angerer et al. in Genetic Engineering: Principles andMethods (Setlow and Hollaender, Eds.) Vol 7, pgs 43-65 (Plenum Press,New York 1985). See also U.S. Pat. Nos. 6,335,167; 6,197,501; 5,830,645;and 5,665,549; the disclosures of which are herein incorporated byreference. Hybridizing the sample to the array is typically performedunder stringent hybridization conditions, as described herein and asknown in the art. Selection of appropriate conditions, includingtemperature, salt concentration, polynucleotide concentration,time(duration) of hybridization, stringency of washing conditions, andthe like will depend on experimental design, including source of sample,identity of capture agents, degree of complementarity expected, etc.,and are within routine experimentation for those of ordinary skill inthe art to which the invention applies.

Following hybridization, the array-surface bound polynucleotides aretypically washed to remove unbound and not tightly bound labeled nucleicacids. Washing may be performed using any convenient washing protocol,where the washing conditions are typically stringent, as describedabove.

Following hybridization and washing, as described above, thehybridization of the labeled target nucleic acids to the capture agentsis then detected using standard techniques of reading the array, i.e.the array is interrogated. Reading the resultant hybridized array may beaccomplished by illuminating the array and reading the location andintensity of resulting fluorescence at each feature of the array todetect any binding complexes on the surface of the array. For example, ascanner may be used for this purpose, which is similar to the AGILENTMICROARRAY SCANNER available from Agilent Technologies, Palo Alto,Calif. Other suitable devices and methods are described in U.S. patentapplications: Ser. No. 09/846,125 “Reading Multi-Featured Arrays” byDorsel et al.; and U.S. Pat. No. 6,406,849. However, arrays may be readby any other method or apparatus than the foregoing, with other readingmethods including other optical techniques (for example, detectingchemiluminescent or electroluminescent labels) or electrical techniques(where each feature is provided with an electrode to detecthybridization at that feature in a manner disclosed in U.S. Pat. No.6,221,583 and elsewhere). In the case of indirect labeling, subsequenttreatment of the array with the appropriate reagents may be employed toenable reading of the array. Some methods of detection, such as surfaceplasmon resonance, do not require any labeling of nucleic acids, and aresuitable for some embodiments.

Results from the reading or evaluating may be raw results (such asfluorescence intensity readings for each feature in one or more colorchannels) or may be processed results (such as those obtained bysubtracting a background measurement, or by rejecting a reading for afeature which is below a predetermined threshold, normalizing theresults, and/or forming conclusions based on the pattern read from thearray (such as whether or not a particular target sequence may have beenpresent in the sample, or whether or not a pattern indicates aparticular condition of an organism from which the sample came).

In certain embodiments, results from interrogating the array are used toassess the level of binding of the population of labeled nucleic acidsto capture agents on the array. The term “level of binding” means anyassessment of binding (e.g. a quantitative or qualitative, relative orabsolute assessment) usually done, as is known in the art, by detectingsignal (i.e., pixel brightness) from a label associated with the samplenucleic acids, e.g. the digested sample is labeled. The level of bindingof labeled nucleic acid to capture agent is typically obtained bymeasuring the surface density of the bound label (or of a signalresulting from the label).

In certain embodiments, a surface-bound polynucleotide may be assessedby evaluating its binding to two populations of nucleic acids that aredistinguishably labeled. In these embodiments, for a singlesurface-bound polynucleotide of interest, the results obtained fromhybridization with a first population of labeled nucleic acids may becompared to results obtained from hybridization with the secondpopulation of nucleic acids, usually after normalization of the data.The results may be expressed using any convenient means, e.g., as anumber or numerical ratio, etc.

EXAMPLES

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of synthetic organic chemistry,biochemistry, molecular biology, and the like, which are within theskill of the art. Such techniques are explained fully in the literature.Unless otherwise defined herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention belongs.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toperform the methods and use the compositions disclosed and claimedherein. Efforts have been made to ensure accuracy with respect tonumbers (e.g., amounts, temperature, etc.) but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C. and pressure is at or nearatmospheric. Standard temperature and pressure are defined as 20° C. and1 atmosphere.

Experimental Methods:

RNA ligation was assessed with synthetic RNA oligonucleotides (21-23nucleotides, Dharmacon) in reaction solutions containing 0, 15, 20, 25,and 30% DMSO. The reactions containing 25% DMSO were assayed with andwithout the pre-heating step. Stock solutions of 20 μM RNAoligonucleotides were stored in 1×TE (10 mM Tris-HCl, pH 7.5, 1 mMEDTA). Initial mixtures of RNA, DMSO and water were first assembled. Forpre-heated samples, the heated mixture contained 40-70% DMSO and wereheated using a 104° C. heating block for 1.5-2 minutes. The heatedsamples were immediately set on ice for >5 minutes prior to finalassembly. The final reaction contains 1×Amersham Pharmacia RNA ligasebuffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 10 mM DTT, 1 mM ATP, 60ng/μL BSA) 1 unit/μL T4 RNA ligase, 100 μM,5′-phosphate-cytidyl-phosphate-Cy5-3′ (pCpCy5) or5′-phosphate-cytidyl-phosphate-Cy3-3′(pCpCy3) (Dharmacon) and 2-4 μM RNAoligonucleotides. The reactions were incubated at 16° C. overnight. RNAligase was inactivated by heating the reaction solutions using a 104° C.heating block for 1.5-2 minutes, followed by immediately setting on icefor >5 minutes.

The labeling efficiency was determined by 5′-phosphorylation of RNAligation reaction aliquots with radioactive P³²-gamma-ATP. The resultingmixture was desalted with Micro Bio-Spin™ (BioRad) desalting columns.The desalted mixture was loaded onto denaturing polyacrylamide gel.Since the ligation products contain an extra nucleotide and fluorophore,they have a lower electrophoretic migration rate than the unligatedprecursors. P³²-labeled RNA bands are visualized and quantified withphosphorimager (Molecular Dynamics). The ligation efficiency wasdetermined by the ratio of ligated vs. unligated P³²-labeled RNA bands.Thus, ligation efficiency may be expressed as the mol % of initial RNAthat winds up having an attached label moiety.

Description of Experiments:

In the experiments described here, T4 RNA ligase is used to labelsynthetic RNA oligonucleotides with5′-phosphate-cytidyl-phosphate-Cy5-3′ (pCpCy5) or5′-phosphate-cytidyl-phosphate-Cy3-3′(pCpCy3). The reaction conditionsdescribed here have been observed to result in ligation efficiencies ofabout 60% or more, e.g. about 70% or more, or 80% or more, up to about95% or more, e.g. up to about 99% with minimal sequence discrimination.This was accomplished by reacting at 25% DMSO, 16° C. overnight, withdonor to acceptor ratio of >12.5:1. The reaction buffer contains 50 mMTris-HCl, pH 7.5, 10 mM MgCl2, 10 mM DTT, 1 mM ATP, 60 μg/mL BSA, and25% DMSO. Typical reactions are 10 μL with 2 μM RNA, 100 μM pCpCy5 orpCpCy3, and 4 units T4 RNA ligase (Amersham/Pharmacia). Reactionefficiency seemed unaffected by increasing the RNA concentration to 8 μMor decreasing enzyme to 2 units.

The labeling efficiency was determined by first performing the ligationreaction. An aliquot of the ligation mixture was then labeled on the 5′end with radioactive P³²-γ-ATP and T4 Polynucleotide Kinase. The controlsample, which did not undergo ligation reaction, and final reactionmixture was denatured with formamide and assessed with denaturingpolyacrylamide gel electrophoresis (1× or 0.5×TBE, 50% urea, 15-20%polyacrylamide with 19:1 acrylamide to bisacrylamide ratio, at about 50°C.). The resulting gel was scanned with Molecular Dynamics StormPhosphorimager for pCpCy5-labeled RNA. The ligation product was clearlyvisible as a red fluorescent miRNA. The gel was then exposed to phosphorscreen to determine the pCpCy5-labeling efficiency. Since the additionof pCpCy5 increases the acceptor miRNA by 1 nucleotide and afluorophore, the mobility of the cy5-labeled strand was lower than theunreacted strand. They appear as distinct bands when scanned in thephosphor mode on the phosphoimager; this was further verified by therelative mobility between the ligase reacted samples and unreactedcontrols (both the ligase reacted samples and the unreacted controlswere labeled with P³²-γ-ATP). Thus the relative level of radioactivitybetween the Cy-labeled and unlabeled bands reveals the ligationefficiency. The reaction efficiency of pCpCy3 was determined similarlyexcept the Cy3 labeled strand was undetectable by the fluorescent modeof the phosphorimager. The product and reactant miRNA bands of the Cy3reaction were defined by the mobility of Cy5 reactions in polyacrylamidegel electrophoreseis and phosphorimager analysis.

Ligation efficiency under different reaction conditions was extensivelytested with pCpCy5 and 4 separate synthetic oligonucleotides (SEQ IDNOs:1-4), each of which contains the same sequence as drosophila miRNA(as indicated): TABLE 1 Test Sample miRNAs SEQ 3′ ID Terminal # NO: NameSequence NT NT 1 dme-miR-3 UCACUGGGCAAAGUGUGUCUCA A 22 2 dme-let-7UGAGGUAGUAGGUUGUAUAGU U 21 3 dme-miR-14 UCAGUCUUUUUCUCUCUCCUA A 21 4dme-miR-31a UGGCAAGAUGUCGGCAUAGCUGA A 23These miRNAs were labeled with 80-99% efficiency when the reactionmixture contained 95% (molar ratio) competitors composing of othermiRNAs and longer single stranded RNAs (100-500 nts). Thus it isreasonable to expect high labeling efficiency in heterogeneousbiological RNA mixtures.

After optimization of labeling efficiencies of these RNAs with Cy5, thelabeling reaction was expanded to include the following sequences (SEQID NOs:6-10) with pCpCy5 and pCpCy3 in separate studies. Theseadditional strands address any bias that may result from 3′ terminalnucleotide, potential secondary structures and nucleotide content of themiRNA. TABLE 2 Additional Test Sample miRNAs 3′ SEQ Termi- ID nal # NO:Name Sequence NT NT 5 dme-miR-2b UAUCACAGCCAGCUUUGAGGAGC C 23 6dme-miR-6 UAUCACAGUGGCUGUUCUUUUU U 22 7 dme-miR-184*CCUUAUCAUUCUCUCGCCCCG G 21 8 dme-miR-285 UAGCACCAUUCGAAAUCAGUGC C 22 9dme-miR-308 AAUCACAGGAUUAUACUGUGAG G 22 10 dme-miR-316UGUCUUUUUCCGCUUACUGGCG G 22

Potentially, the RNA ligase method can be used for dyes other than Cy5and Cy3, but the efficiency may differ from the ones presented here.Moreover, it is possible to determine the labeling efficiencies of eachindividual miRNA of a given set and perform highly quantitativemicroarray experiments by correlating fluorophore counts with number ofmolecule. For example, in an array hybridization experiment wherein anarray is contacted with a labeled RNA sample, it is possible toascertain the total quantity of fluorophores in a given area of thearray by interrogating (or scanning) the array; given the labelingefficiency of the labeled RNA sample (determined as disclosed herein),the quantity of RNA hybridized to the given area of the array may bedetermined.

Given that the approximate labeling efficiency may be determined (asdescribed herein), in particular embodiments the present invention thusprovides quantitative methods of performing array hybridizationexperiments. It is expected that this will provide a more sensitiveassay system for the detection of variations of miRNA, such as found indevelopmental stages, tissue samples, disease states, as well as anyindividual and/or abnormal variations. Moreover, if more viral miRNAsare identified, this can become a novel diagnostic tool for active aswell as latent viral infections

Determination of Labeling Efficiency of miRNAs in Complex Samples

RNA ligase is used to label a complex RNA mixture, such as the total RNAor isolated mixtures of small RNAs from biological samples. The labeledmixtures are run on denaturing polyacrylamide gel and Northern blots areperformed of individual miRNAs with radioactive probes. The RNAs labeledby RNA ligase will have a lower mobility relative to its unlabeledcounterpart. Thus each target sequence will run as a doublet when probedby Northern blot. The ratio of RNA species in these doublets reflectsthe molar ratio of the RNA ligase labeled vs. unlabeled RNA species.

Microarray Hybridization:

The synthetic miRNA set forth above were either labeled with Cy5 or Cy3and hybridized onto microarrays as follows:

Labeled miRNA were desalted with BioRad Micro Bio-Spin™ 6 (as directedby BioRad instructions) to remove free fluorescent tags. The desaltedmiRNA was added to solution containing water and carrier (25-mer DNAwith random sequence). The solution was heated for approximately 1minute per 10 ul solution at 100° C. and immediately placed on ice.After cooling, 2×Agilent Hyb Buffer (1225 mM LiCl, 300 mM Li-MES, pH6.1, 12 mM EDTA, 3.0% (w/v) lithium dodecyl sulfate, 2.0% (w/v) TritonX-100) was added to the mixture and the viscous liquid was mixedcarefully. The final solution contained 1×Hyb Buffer and 0.1 μg/μlrandom 25-mer. The concentration of miRNA was varied for differentexperiments.

Hybridization was performed with SureHyb hybridization chamber (AgilentPart Number: G2534A) and place on rotisserie of hybridization ovenovernight. The hybridization temperature was tested at 50° C. and 60° C.

After hybridization was completed, the Sure-Hyb chamber complex wasremoved from the oven and immediately disassembled in Wash Buffer 1(6×SSC, 0.005% Triton X-102) at room temperature. The microarray wastransferred to a fresh wash chamber containing Wash Buffer 1 and washedby stirring for 10 minutes at room temperature. The microarray was thenwashed in Wash Buffer 2 (1.1×SSC, 0.005% Triton X-102) by stirring atroom temperature for 5 minutes. The microarray was slowly lifted out ofthe wash chamber after washing and dried with nitrogen as needed. Themicroarrays were scanned with Agilent Scanner (Agilent Product Number:G2565BA). The scanned data was extracted with Agilent Feature ExtractionSoftware (Agilent Product Number; G2567AA) and the green and redbackground-subtracted signals were evaluated for hybridizationefficiency and specificity. Data was further analyzed using Spotfiresoftware and Microsoft Excel.

While the foregoing embodiments of the invention have been set forth inconsiderable detail for the purpose of making a complete disclosure ofthe invention, it will be apparent to those of skill in the art thatnumerous changes may be made in such details without departing from thespirit and the principles of the invention. Accordingly, the inventionshould be limited only by the following claims.

All patents, patent applications, and publications mentioned herein arehereby incorporated by reference in their entireties, provided that, ifthere is a conflict in definitions, the definitions provided hereinshall control.

1. A method of labeling RNA in a sample, the method comprising:contacting the sample with an enzyme having an RNA ligation activity inthe presence of a labeled substrate under conditions sufficient toresult in coupling of the labeled substrate to the RNA in the sample toprovide labeled RNA, the conditions including a DMSO concentration inthe range from about 20% to about 30%, wherein the labeled substratecomprises an observable label moiety attached to a nucleotide moiety. 2.The method of claim 1, wherein the nucleotide moiety is selected from amononucleotide moiety or an oligonucleotide moiety.
 3. The method ofclaim 1, wherein the nucleotide moiety has a length in the range from 2to about 50 bases.
 4. The method of claim 1, wherein the RNA in thesample comprises isolated RNA having length less than about 500 bases.5. The method of claim 1, wherein the RNA in the sample comprisesisolated RNA having length less than about 200 bases.
 6. The method ofclaim 1, wherein the labeled substrate has the structureN-Dwherein N is selected from a mononucleotide moiety or anoligonucleotide moiety having a length of less than about 100 bases, andD is an observable label moiety attached to N.
 7. The method of claim 6,wherein N has a 5′ terminus, a 3′ terminus, and an internal attachmentsite, wherein D is attached to N via a site selected from the 5′terminus, the 3′ terminus, and the internal attachment site.
 8. Themethod of claim 1, wherein the observable label moiety is selected froma chromogenic moiety, a fluorophore, a mass label, a spin label, or aradiolabel.
 9. The method of claim 1, wherein the observable labelmoiety is a fluorophore selected from the group consisting Cy3, Cy5, oran Alexa dye.
 10. The method of claim 1, wherein the nucleotide moietyhas a free 5′-phosphate and a 3′ terminus bound to the observable labelmoiety.
 11. The method of claim 1, wherein the nucleotide moiety has afree 3′-OH and a 5′ terminus bound to the observable label moiety. 12.The method of claim 1, wherein the labeled substrate comprises aplurality of observable label moieties and the labeled RNA consistsessentially of RNA labeled with a plurality of label moieties.
 13. Themethod of claim 1, wherein the labeled substrate comprises a singleobservable label moiety and the labeled RNA consists essentially ofsingly-labeled RNA.
 14. The method of claim 1, wherein the labeled RNAis at least 70% of the initial RNA in the sample.
 15. The method ofclaim 1, further comprising, prior to the contacting, heating the sampleto at least about 80° C. under conditions including at least about 40%DMSO.
 16. The method of claim 15, wherein the duration of heating is atleast 10 seconds.
 17. The method of claim 1, further comprising, priorto contacting the sample with the enzyme having an RNA ligationactivity, contacting the sample with an enzyme having a 5′-phosphataseactivity to remove 5′-phosphate groups from the RNA in the sample. 18.The method of claim 1, wherein the enzyme is capable of coupling aspecies selected from the group consisting of a nucleotide having a 5′phosphate, an oligonucleotide having a 5′ phosphate, and an RNA having a5′ phosphate to an oligonucleotide having a 3′ hydroxyl.
 19. The methodof claim 1, wherein the enzyme is selected from T4 RNA ligase, RNAligase II, yeast poly A polymerase, E. coli poly A polymerase, orterminal transferase.
 20. The method of claim 1, wherein the enzyme isT4 RNA ligase.
 21. A method of performing an array analysis comprising:labeling RNA in a sample using a method according to claim 1 to providethe labeled RNA; contacting the labeled RNA with an array underconditions sufficient to provide for specific binding of labeled RNA tothe array; and interrogating the array to provide data on binding of thelabeled RNA to the array.